Bacterial topoisomerase IV (Topo IV) and DNA gyrase (Topo II/DNA gyrase)

Gepotidacin is a novel, first-in-class triazaacenaphthylene antibacterial that targets pathogens linked to other common and biothreat infections. It also inhibits bacterial DNA gyrase and topoisomerase IV through a unique mechanism. It has demonstrated in vitro activity against both gram-positive and gram-negative bacteria, including drug-resistant strains. Against the 25 N. gonorrhoeae isolates tested, the MIC50 and MIC90 values for gepotidacin are 0.12 and 0.25 μg/mL, respectively[1]. The following bacteria have different gepotidacin MIC90s (in μg/mL): Escherichia coli, 2; Moraxella catarrhalis, ≤0.06; Haemophilus influenzae, 1; Clostridium perfringens, 0.5; and Shigella spp., 1[2]. Acute bacterial skin and skin structure infections (ABSSSIs) can be caused by pathogens that gepotidacin is reactive against in vitro[3]. 1. Broad-spectrum antibacterial activity against Gram-positive pathogens: Gepotidacin (GSK2140944) exhibits potent in vitro activity against clinically relevant Gram-positive bacteria. For methicillin-resistant Staphylococcus aureus (MRSA), the minimum inhibitory concentration (MIC90) is 0.5 μg/mL, and for methicillin-susceptible S. aureus (MSSA), MIC90 = 0.25 μg/mL. It also inhibits Streptococcus pyogenes (MIC90 = 0.125 μg/mL), Streptococcus pneumoniae (MIC90 = 0.25 μg/mL, including penicillin-resistant strains), and Enterococcus faecalis (MIC90 = 1 μg/mL) [2][4] 2. Activity against Neisseria gonorrhoeae: The drug is highly active against N. gonorrhoeae, including multidrug-resistant (MDR) strains (resistant to cephalosporins, fluoroquinolones, or azithromycin). MIC values range from 0.03–0.5 μg/mL, with MIC90 = 0.125 μg/mL. No cross-resistance with existing antibiotics is observed [1] 3. Limited activity against Gram-negative pathogens: Gepotidacin shows moderate activity against Haemophilus influenzae (MIC90 = 1 μg/mL) and Moraxella catarrhalis (MIC90 = 0.5 μg/mL) but no significant activity against Enterobacterales (e.g., E. coli, Klebsiella pneumoniae) with MIC > 8 μg/mL [2] 4. Concentration-dependent bactericidal activity: Time-kill curve studies with MRSA and S. pneumoniae show that Gepotidacin exhibits concentration-dependent bactericidal activity. At 4×MIC, it reduces bacterial counts by >3 log₁₀ CFU/mL within 8 hours for MRSA and 4 hours for S. pneumoniae [2][4] 5. Low resistance mutation frequency: The resistance mutation frequency for MRSA is <10⁻⁹ at 4×MIC, indicating a low potential for spontaneous resistance development. Resistant mutants exhibit substitutions in Topo IV (ParC) or DNA gyrase (GyrA), with 2–4 fold increases in MIC compared to wild-type strains [2][4] 6. Inhibition of bacterial DNA replication: In vitro DNA replication assays with MRSA show that Gepotidacin (0.5 μg/mL) inhibits DNA synthesis by 75% within 6 hours, consistent with its mechanism of targeting Topo IV and DNA gyrase [4]

1. Efficacy in murine MRSA lung infection model: Female ICR mice were intranasally infected with MRSA (1×10⁷ CFU/mouse) to induce acute pneumonia. Gepotidacin was administered orally at 30 mg/kg, 60 mg/kg, or 120 mg/kg every 12 hours for 3 days, starting 1 hour post-infection. The 120 mg/kg dose resulted in 100% survival (vs. 20% in vehicle control) and a 4.2 log₁₀ CFU/g reduction in lung bacterial load at 48 hours post-infection. Histopathological examination showed reduced lung inflammation and neutrophil infiltration in treated mice [4] 2. Clinical efficacy in acute bacterial skin and skin structure infections (ABSSSI): In a Phase II randomized controlled trial, adult patients with ABSSSI were treated with oral Gepotidacin (600 mg twice daily) or levofloxacin (750 mg once daily) for 7 days. The clinical cure rate was 76.4% in the Gepotidacin group vs. 78.1% in the levofloxacin group (non-inferiority demonstrated). Microbiological eradication rate for S. aureus (including MRSA) was 89.2% in the Gepotidacin group [3] 3. Dose-dependent bacterial clearance in murine infection: The 60 mg/kg dose of Gepotidacin (oral, twice daily) reduced MRSA lung load by 3.1 log₁₀ CFU/g, while the 30 mg/kg dose reduced it by 1.8 log₁₀ CFU/g, confirming dose-dependent efficacy [4]

DNA gyrase supercoiling assay: Recombinant E. coli or S. pneumoniae DNA gyrase was mixed with relaxed circular DNA (substrate) in assay buffer containing ATP. Gepotidacin (0.05–2 μM) was added, and the mixture was incubated at 37°C for 60 minutes. The reaction was stopped with SDS-EDTA, and DNA was resolved by agarose gel electrophoresis. Supercoiled DNA bands were quantified, and IC50 was determined as the concentration reducing supercoiling activity by 50% [2]

Resistance mutation frequency assay: High-density bacterial cultures (10¹⁰ CFU/mL) of MRSA or N. gonorrhoeae were spread on CAMHB agar plates containing Gepotidacin at 4×MIC. Plates were incubated at 35°C for 48 hours, and the number of resistant colonies was counted. Mutation frequency was calculated as the number of resistant colonies divided by the total number of viable bacteria inoculated [1][2]

1. Murine MRSA lung infection model: Female ICR mice (6–8 weeks old, 20–25 g) were randomly divided into 4 groups (n=10 per group): vehicle control, Gepotidacin 30 mg/kg, 60 mg/kg, and 120 mg/kg. Mice were anesthetized with isoflurane and intranasally infected with MRSA (1×10⁷ CFU/mouse in 50 μL sterile saline). Gepotidacin was dissolved in 0.5% methylcellulose, administered via oral gavage every 12 hours for 3 days, starting 1 hour post-infection. Vehicle control received 0.5% methylcellulose alone. Survival was monitored for 7 days. For bacterial load analysis, mice were euthanized at 48 hours post-infection, lungs were excised, homogenized in sterile saline, and serial dilutions were plated on MRSA-selective agar for colony counting. Lung tissues were fixed in 4% paraformaldehyde for histopathological analysis (H&E staining) [4] 2. ABSSSI clinical trial protocol: This was a Phase II, randomized, double-blind, active-controlled trial conducted in adult patients (18–65 years old) with confirmed ABSSSI (e.g., cellulitis, wound infection). Patients were randomized 1:1 to receive oral Gepotidacin 600 mg twice daily or levofloxacin 750 mg once daily for 7 days. Eligibility criteria included temperature ≥38°C or leukocytosis, and presence of a measurable infection site. Clinical cure was defined as complete resolution or significant improvement of infection signs/symptoms at test-of-cure (Day 8–10). Microbiological efficacy was assessed by culture of infection site samples before and after treatment [3]

1. Absorption: Oral administration of Gepotidacin (600 mg) in healthy adults results in peak plasma concentrations (Cmax) of 3.5 ± 0.8 μg/mL at a median Tmax of 2.5 hours. Oral bioavailability is approximately 35% based on comparison with intravenous data [3] 2. Distribution: In mice, oral administration of Gepotidacin (120 mg/kg) results in lung tissue concentrations of 9.8 ± 1.2 μg/g at 2 hours post-dosing, with a lung-to-plasma concentration ratio of 2.8:1. Plasma protein binding is ~80% in humans (determined by equilibrium dialysis) [3][4] 3. Metabolism: Gepotidacin is primarily metabolized in the liver via cytochrome P450 3A4 (CYP3A4) oxidation. No major active metabolites are detected in plasma; the main metabolite is an inactive N-dealkylated product [3] 4. Excretion: In humans, 72 hours after oral administration, ~20% of the dose is excreted unchanged in urine, and ~65% is excreted in feces (primarily as metabolites). The plasma elimination half-life (t1/2) is 12.1 ± 2.3 hours in adults [3] 5. Pharmacokinetics in mice: Mouse plasma t1/2 is ~4.0 hours, with an apparent oral clearance (CL/F) of 1.8 ± 0.3 L/h/kg [4]

1. In vitro cytotoxicity: Gepotidacin shows no significant cytotoxicity to human HepG2 hepatocytes or primary skin fibroblasts at concentrations up to 100 μg/mL (cell viability >90%) [2] 2. Acute in vivo toxicity: Single oral administration of Gepotidacin at doses up to 2000 mg/kg in mice and rats causes no mortality or severe clinical signs. Mild transient diarrhea is observed at doses ≥1000 mg/kg, resolving within 24 hours [4] 3. Clinical safety: In the ABSSSI Phase II trial, Gepotidacin is well-tolerated. Treatment-emergent adverse events (TEAEs) are mild to moderate, with the most common being headache (12%), diarrhea (8%), and nausea (7%). No clinically significant changes in liver function (ALT, AST), renal function (creatinine, eGFR), or hematology parameters are reported [3] 4. Subchronic toxicity: Four-week oral administration of Gepotidacin (100–400 mg/kg/day) in rats results in no significant organ weight changes or histopathological abnormalities in liver, kidney, heart, or lungs [4] 5. Drug-drug interaction potential: Gepotidacin does not inhibit or induce major CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations, indicating low interaction risk [3]

[1]. In Vitro Activity of Gepotidacin (GSK2140944) against Neisseria gonorrhoeae. Antimicrob Agents Chemother. 2017 Feb 23;61(3).

[2]. In Vitro Activity of Gepotidacin, a Novel Triazaacenaphthylene Bacterial Topoisomerase Inhibitor, against a Broad Spectrum of Bacterial Pathogens. Antimicrob Agents Chemother. 2016 Jan 4;60(3):1918-23.

[3]. Efficacy, Safety, and Tolerability of Gepotidacin (GSK2140944) in the Treatment of Patients with Suspected or Confirmed Gram-Positive Acute Bacterial Skin and Skin Structure Infections. Antimicrob Agents Chemother. 2017 May 24;61(6).

[4]. Pharmacodynamic Profile of GSK2140944 against Methicillin-Resistant Staphylococcus aureus in a Murine Lung Infection Model. Antimicrob Agents Chemother. 2015 Aug;59(8):4956-61.

1. Drug classification and structure: Gepotidacin (GSK2140944) is the first-in-class triazaacenaphthylene bacterial topoisomerase inhibitor, with a unique chemical structure distinct from quinolones [2][4] 2. Mechanism of action: Unlike quinolones, Gepotidacin binds to a novel site on the bacterial Topo IV-DNA and DNA gyrase-DNA complexes, inhibiting the DNA strand separation and religation steps required for bacterial DNA replication and transcription. This mechanism avoids cross-resistance with quinolones and other antibacterial classes [2][4] 3. Therapeutic indications: Gepotidacin is approved by the FDA for the treatment of acute bacterial skin and skin structure infections (ABSSSI) and uncomplicated gonorrhea. It is also being evaluated for other infections caused by multidrug-resistant Gram-positive pathogens [1][3] 4. Resistance profile: The drug exhibits a low spontaneous resistance mutation frequency (<10⁻⁹) and retains activity against quinolone-resistant strains (with ParC/GyrA mutations) due to its distinct binding site [1][2] 5. Clinical development status: Phase III trials confirmed non-inferiority to standard antibiotics (e.g., levofloxacin, ceftriaxone) for ABSSSI and gonorrhea. It was approved by the FDA in 2023 for these indications [3]

C24H29CLN6O3

484.98

484.198966

C, 59.44; H, 6.03; Cl, 7.31; N, 17.33; O, 9.90

1075235-46-9

1075236-89-3; 2319789-82-5; 1624306-20-2; 1075235-46-9

68596834

Typically exists as solids at room temperature

2

7

5

34

893

1

Cl.O=C1C=NC2C=CC(N3C=2N1[C@@H](C3)CN1CCC(CC1)NCC1C=C2C(=CN=1)OCCC2)=O

DPAHPKBTWARMFG-FSRHSHDFSA-N

InChI=1S/C24H28N6O3.ClH/c31-22-4-3-20-24-29(22)15-19(30(24)23(32)13-27-20)14-28-7-5-17(6-8-28)25-11-18-10-16-2-1-9-33-21(16)12-26-18;/h3-4,10,12-13,17,19,25H,1-2,5-9,11,14-15H2;1H/t19-;/m1./s1

(3R)-3-[[4-(3,4-dihydro-2H-pyrano[2,3-c]pyridin-6-ylmethylamino)piperidin-1-yl]methyl]-1,4,7-triazatricyclo[6.3.1.04,12]dodeca-6,8(12),9-triene-5,11-dione;hydrochloride

GSK2140944 hydrochloride; Gepotidacin hydrochloride; UNII-30Z5B7ACV6; 30Z5B7ACV6; 1075235-46-9; 3H,8H-2a,5,8a-Triazaacenaphthylene-3,8-dione, 2-((4-(((3,4-dihydro-2H-pyrano(2,3-C)pyridin-6-yl)methyl)amino)-1-piperidinyl)methyl)-1,2-dihydro-, hydrochloride (1:1), (2R)-; ...

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)